The biological activities of the neem tree seeds have long been recognized. Of primary importance are the potent insecticidal and pesticidal properties of azadirachtin, the main active ingredient in the neem seed. Azadirachtin is a tetranortriterpenoid that causes feeding inhibition and growth disruption in various insect, mite, nematode, etc. orders.
There are various methods known to extract azadirachtin from neem seeds. Typically, these methods involve drying the neem seeds, milling the dried seeds to a coarse powder, and extracting the powder with various solvents such as methanol, ethanol, water, methylene chloride, chloroform, hexane methylethylketone, butanol, petroleum benzene, ether, acetone, methyl tertbutyl ether, diethylcarbonate, etc. In general, it has been found that the efficiency of the extract yield can be increased by increasing the solvent polarity, i.e., from hexane to ethanol, ethanol to methanol, etc. However, despite the initial drying of the neem seeds, they still may contain between 6 to 15% water. Thus, while the utilization of more polar solvents will increase the extraction efficiency relative to azadirachtin, it will also extract more of the water contained within the neem seeds and results in aqueous-containing extracts. That is, since water and azadirachtin have similar solubilities, the solvents useful for extracting the azadirachtin from neem seeds also extract water contained therein. Crude neem seed extracts may contain up to 20% water.
Early study of the bioactive properties of neem seeds was directed to identifying fractions, including oil and wax fractions that retained bioactivity after preparation. See, e.g., Singh, et al., Phytoparasitica, 16:3, 1988. U.S. Pat. No. 4,556,562 (Larson) teaches a storage-stable, acidified aqueous ethanol extract of azadirachtin prepared from ground neem seeds. An accepted method for preparing azadirachtin includes extracting oil from the seeds with an azadirachtin-insoluble solvent such as hexane, followed by a second extraction using an azadirachtin-soluble solvent such as ethanol to obtain an azadirachtin-containing extract solution. However, U.S. Pat. No. 4,946,681 (Walter) teaches that protic solvents having acidic or basic functional groups decrease the storage stability of azadirachtin. U.S. Pat. No. 4,946,681 teaches a method for using molecular sieves to remove water from an extract solution without removing azadirachtin, to improve the storage stability of the extract. The same patent also teaches that the storage stability of azadirachtin in an extract solution varies greatly, depending upon the solvent system of the extract. Storage stability is enhanced when the extract solution comprises greater than 50% by volume aprotic solvents and less than 15% water. Aprotic solvents are defined in U.S. Pat. No. 4,946,681 as polar solvents having moderately high dielectric constants which do not contain acidic hydrogen, including, but not limited to, aliphatic alcohols, ketones, nitriles, substituted aromatics, amide sulfoxides, alkyl carbonates, chlorinated aliphatics, aromatic aldehydes, sulfones, ethers, esters, and the like, or mixtures thereof. U.S. Pat. No. 5,001,146 (Carter, et al.) teaches storage of azadirachtin in non-degrading solvent systems that offer improved shelf-life stability over ethanol-water based formulations. U.S. Pat. No. 5,001,146 also teaches that if the protic solvent is an alcohol, the concentration of water must be less than 5% water by volume, and is preferably less than 2%, and most preferably less than 1% of the total solution.
At present, all commercial preparations comprising azadirachtin are organic solutions. The commercial use of azadirachtin has been limited as a result of storage instability of the compound. Water has been identified as the primary cause of degradation of azadirachtin in solution.
Several methods are known for preparing solid azadirachtin. U.S. Pat. No. 5,397,571 (Roland, et al.) describes a method for co-extracting azadirachtin and neem oil from ground neem seeds. One product of the method of U.S. Pat. No. 5,397,571 is an azadirachtin-containing solid having greater than 10 weight percent of azadirachtin. Briefly, in this method, ground neem seeds are co-extracted with a mixture of nonpolar, aliphatic hydrocarbon solvents and polar solvents, each present in an amount sufficient to permit extraction of both the hydrophilic and hydrophobic components desired from the neem seeds. The hydrophilic and hydrophobic portions of the solvent extract mixture are separated from each other, preferably using a counter-extraction process, whereby the solvent is removed from the solvent extract mixture to leave a neem extract mixture that contains both the hydrophilic and the hydrophobic portions. The neem extract mixture is then contacted with a solvent having a sufficiently low polarity (i.e., hexane) to precipitate as a solid the hydrophilic, azadirachtin-containing portion of the extract. The solid is then recovered by a known technique, such as filtration.
In the method of Schroeder, D. R. and K. Nakanishi, J. Natural Products 50:241-244 (1987), neem seeds are exhaustively defatted with hexane and are then extracted with 95% ethanol/5% water. The ethanol extract is then partitioned between petroleum ether and 95% aqueous methanol. The ether phase, containing oils and non-polar materials, is discarded. The methanol phase is then partitioned between water and ethyl acetate. The water phase is discarded and the acetate phase is vacuum-filtered through a plug of Si gel and then concentrated. The concentrated filtrate is subjected to vacuum liquid chromatography in hexane-ethyl acetate (1:3). The azadirachtin-containing fractions (70-80% pure) are combined and azadirachtin is crystallized using carbon tetrachloride. Final purification is by flash chromatography in CHCl.sub.3 --MeCN, 3:1.
Existing azadirachtin-containing solids, while offering advantages over liquid preparations, are unstable and have unique storage requirements. Schroeder and Nakanishi recognized the instability of their solid product, noting that the material has a half-life of about 4 months in the solid state in flasks at room temperature. Id. at 243, note 3.
It would be desirable to provide a storage-stable, azadirachtin-containing solid that could be combined with diluents, surfactants or dispersants to produce a storage-stable biocontrol agent.